Reactive oxygen intermediates (ROI) are cytotoxic and mutagenic. They modify and damage critical biomolecules including DNA and lipids. The partial reduction products of oxygen are: 1 electron reduces O2 to form superoxide (O2−), and 2 electrons reduce O2 to form hydrogen peroxide (H2O2). The cytotoxic property of ROI is exploited by phagocytes, which generate large amounts of superoxide and hydrogen peroxide as part of their armory of bactericidal mechanisms. ROI have been considered an accidental byproduct of metabolism, particularly mitochondrial respiration. However, recent studies give evidence for regulated enzymatic generation of O2− and its conversion to H2O2 in a variety of cells. The conversion of O2− to H2O2 can also occur spontaneously, but is markedly accelerated by superoxide dismutase (SOD). Exposure of cells to platelet derived growth factor and epidermal growth factor induces the production of H2O2, which activates components of signaling pathways including p42/p44 MAPK and tyrosine phosphorylation.
Several biological systems generate reactive oxygen. Exposure of neutrophils to bacteria or to various soluble mediators such as formyl-Met-Leu-Phe or phorbol esters activates a massive consumption of oxygen, termed the respiratory burst, to initially generate superoxide, with secondary generation of H2O2, HOCl and hydroxyl radicals. The enzyme responsible for this oxygen consumption is the respiratory burst oxidase (nicotinamide adenine dinucleotide phosphate-reduced form (NADPH) oxidase).
There is growing evidence for the generation of ROI by non-phagocytic cells, particularly in situations related to cell proliferation. Significant generation of H2O2, O2−, or both have been noted in some cell types. Fibroblasts and human endothelial cells show increased release of superoxide in response to cytokines such as interleukin-1 or tumor necrosis factor (TNF) (Meier et al. (1989) Biochem J. 263, 539–545; Matsubara et al. (1986) J. Immun. 137, 3295–3298). Ras-transformed fibroblasts show increased superoxide release compared with control fibroblasts (Irani, et al. (1997) Science 275, 1649–1652). Rat vascular smooth muscle cells show increased H2O2 release in response to PDGF (Sundaresan et al. (1995) Science 270, 296–299) and angiotensin II (Griendling et al. (1994) Circ. Res. 74, 1141–1148; Fukui et al. (1997) Circ. Res. 80, 45–51; Ushio-Fukai et al. (1996) J. Biol. Chem. 271, 23317–23321), and H2O2 in these cells is associated with increased proliferation rate. H2O2 in the transformed fibroblasts and in vascular smooth muscle cells is associated with an increased proliferation rate. The occurrence of ROI in a variety of cell types is summarized in Table 1 (adapted from Burdon, R. (1995) Free Radical Biol. Med. 18, 775–794).
TABLE 1SuperoxideHydrogen Peroxidehuman fibroblastsBalb/3T3 cellshuman endothelial cellsrat pancreatic islet cellshuman/rat smooth muscle cellsmurine keratinocyteshuman fat cellsrabbit chondrocyteshuman osteocyteshuman tumor cellsBHK-21 cellsfat cells, 3T3 L1 cellshuman colonic epithelial cells
ROI generated by neutrophils have a cytotoxic function. While ROI are normally directed at the invading microbe, ROI can also induce tissue damage (e.g., in inflammatory conditions such as arthritis, shock, lung disease, and inflammatory bowel disease) or may be involved in tumor initiation or promotion, due to damaging effects on DNA. Nathan (Szatrowski et al. (1991) Canc. Res. 51, 794–798) proposed that the generation of ROI in tumor cells may contribute to the hypermutability seen in tumors, and may therefore contribute to tumor heterogeneity, invasion and metastasis.
In addition to cytotoxic and mutagenic roles, ROI have ideal properties as signal molecules: 1) they are generated in a controlled manner in response to upstream signals; 2) the signal can be terminated by rapid metabolism of O2− and H2O2 by SOD and catalase/peroxidases; 3) they elicit downstream effects on target molecules, e.g., redox-sensitive regulatory proteins such as NFκ-B and AP-1 (Schreck et al. (1991) EMBO J. 10, 2247–2258; Schmidt et al. (1995) Chemistry & Biology 2, 13–22). Oxidants such as O2− and H2O2 have a relatively well defined signaling role in bacteria, operating via the SoxI/II regulon to regulate transcription.
ROI appear to have a direct role in regulating cell division, and may function as mitogenic signals in pathological conditions related to growth. These conditions include cancer and cardiovascular disease. O2− is generated in endothelial cells in response to cytokines, and might play a role in angiogenesis (Matsubara et al. (1986) J. Immun. 137, 3295–3298). O2− and H2O2 are also proposed to function as “life-signals”, preventing cells from undergoing apoptosis (Matsubara et al. (1986) J. Immun. 137, 3295–3298). As discussed above, many cells respond to growth factors (e.g., platelet derived growth factor (PDGF), epidermal derived growth factor (EGF), angiotensin II, and various cytokines) with both increased production of O2−/H2O2 and increased proliferation. Inhibition of ROI generation prevents the mitogenic response. Exposure to exogenously generated O2− and H2O2 results in an increase in cell proliferation. A partial list of responsive cell types is shown below in Table 2 (adapted from Burdon, R. (1995) Free Radical Biol. Med. 18, 775–794).
TABLE 2SuperoxideHydrogen peroxidehuman, hamster fibroblastsmouse osteoblastic cellsBalb/3T3 cellsBalb/3T3 cellshuman histiocytic leukemiarat, hamster fibroblastsmouse epidermal cellshuman smooth muscle cellsrat colonic epithelial cellsrat vascular smooth muscle cellsrat vascular smooth muscle cells
While non-transformed cells can respond to growth factors and cytokines with the production of ROI, tumor cells appear to produce ROI in an uncontrolled manner. A series of human tumor cells produced large amounts of hydrogen peroxide compared with non-tumor cells (Szatrowski et al. (1991) Canc. Res. 51, 794–798). Ras-transformed NIH 3T3 cells generated elevated amounts of superoxide, and inhibition of superoxide generation by several mechanisms resulted in a reversion to a “normal” growth phenotype.
O2− has been implicated in maintenance of the transformed phenotype in cancer cells including melanoma, breast carcinoma, fibrosarcoma, and virally transformed tumor cells. Decreased levels of the manganese form of SOD (MnSOD) have been measured in cancer cells and in vitro-transformed cell lines, predicting increased O2− levels (Burdon, R. (1995) Free Radical Biol. Med. 18, 775–794). MnSOD is encoded on chromosome 6q25 which is very often lost in melanoma. Overexpression of MnSOD in melanoma and other cancer cells (Church et al. (1993) Proc. of Natl. Acad. Sci. 90, 3113–3117; Fernandez-Pol et al. (1982) Canc. Res. 42, 609–617; Yan et al. (1996) Canc. Res. 56, 2864–2871) resulted in suppression of the transformed phenotype.
ROI are implicated in the growth of vascular smooth muscle associated with hypertension, atherosclerosis, and restenosis after angioplasty. O2− generation is seen in rabbit aortic adventitia (Pagano et al. (1997) Proc. Natl. Acad. Sci. 94, 14483–14488). Vascular endothelial cells release O2− in response to cytokines (Matsubara et al. (1986) J. Immun. 137, 3295–3298). O2− is also generated by aortic smooth muscle cells in culture, and increased O2− generation is stimulated by angiotensin II which also induces cell hypertrophy. In a rat model system, infusion of angiotensin II leads to hypertension as well as increased O2− generation in subsequently isolated aortic tissue (Ushio-Fukai et al. (1996) J. Biol. Chem. 271, 23317–23321; Yu et al. (1997) J. Biol. Chem. 272, 27288–27294). Intravenous infusion of a form of SOD that localizes to the vasculature or an infusion of an O2− scavenger prevented angiotensin II induced hypertension and inhibited ROI generation (Fukui et al. (1997) Circ. Res. 80, 45–51).
The neutrophil NADPH oxidase, also known as phagocyte respiratory burst oxidase, provides a paradigm for the study of the specialized enzymatic ROI-generating system. This extensively studied enzyme oxidizes NADPH and reduces oxygen to form O2−. NADPH oxidase consists of multiple proteins and is regulated by assembly of cytosolic and membrane components. The catalytic moiety consists of flavocytochrome b558, an integral plasma membrane enzyme comprised of two components: gp91phox (gp refers to glycoprotein; phox is an abbreviation of the words phagocyte and oxidase) and p22phox (p refers to protein). gp91phox contains 1 flavin adenine dinucleotide (FAD) and 2 hemes as well as the NADPH binding site. p22phox has a C-terminal proline-rich sequence which serves as a binding site for cytosolic regulatory proteins. The two cytochrome subunits, gp91phox and p22phox appear to stabilize one another, since the genetic absence of either subunit, as in the inherited disorder chronic granulomatous disease (CGD), results in the absence of the partner subunit (Yu et al. (1997) J. Biol. Chem. 272, 27288–27294). Essential cytosolic proteins include p47phox, p67phox and the small GTPase Rac, of which there are two isoforms. p47phox and p67phox both contain SH3 regions and proline-rich regions which participate in protein interactions governing assembly of the oxidase components during activation. The neutrophil enzyme is regulated in response to bacterial phagocytosis or chemotactic signals by phosphorylation of p47phox, and perhaps other components, as well as by guanine nucleotide exchange to activate the GTP-binding protein Rac.
ROI generated in many non-phagocytic tissues are now thought to originate from Nox enzymes. These Nox enzymes are homologs of gp91phox, the catalytic subunit of the phagocyte NADPH oxidase. The Nox family consists in human of seven unique gene products: Nox1, Nox2 (same as gp91phox), Nox3, Nox4, Nox5, Duox1 and Duox2. Each member of the Nox family has a specific expression pattern in tissues. For example, Nox1 is highly expressed in colonic epithelium, while Nox4 is highly expressed in kidney epithelium. Although these enzymes are thought to account for much of the ROI generated in many of these tissues, except for Nox2, the mechanism by which these enzymes are regulated is unknown. While not wishing to be bound to any particular theory, it is believed that the molecular candidates related to gp91phox and involved in ROI generation in cells have been located in the Nox and Duox family of proteins. (Lambeth et al. (2001) Gene May 16; 269 (1–2):131–40; Edens et al. (2001) J. Cell Biol. August 20: 154(4):879–91; Lambeth et al. (2000) Trends Biochem Sci. October 25, (10); 459–61)
However, regulatory proteins for this family of enzymes have not been determined. This deficiency has blocked the development of an in vitro system in which the enzymatic activity to generate ROIs can be analyzed. Furthermore, the regulatory proteins would provide an additional target for controlling ROI generation. Accordingly, what is needed is the identity of the regulators of proteins involved in ROI generation, particularly in non-phagocytic tissues and cells. What is also needed are the nucleotide sequences encoding for these proteins, and the primary sequences of the proteins themselves. Also needed are vectors designed to include nucleotides encoding for these proteins. Probes and PCR primers derived from the nucleotide sequence are needed to detect, localize and measure nucleotide sequences, including mRNA, involved in the synthesis of these proteins. In addition, what is needed is a means to transfect cells with these vectors. What is also needed are expression systems for production of these molecules. Also needed are antibodies directed against these molecules for a variety of uses including localization, detection, measurement and passive immunization.